Bioorganic & Medicinal Chemistry 15 (2007) 3201–3207

Anticancer, neuroprotective activities and computational studies

of 2-amino-1,3,4-thiadiazole based compound
Wojciech Rzeski,a,c Joanna Matysiakb,* and Martyna Kandefer-Szerszeńa
a
Department of Virology and Immunology, Institute of Microbiology and Biotechnology,
Maria Curie-Skłodowska University, Lublin, Poland
b
Department of Chemistry, Agricultural University, Lublin, Poland
c
Department of Toxicology, Institute of Agricultural Medicine, Lublin, Poland
Received 28 April 2006; revised 15 February 2007; accepted 20 February 2007
Available online 22 February 2007

Abstract—Anticancer activity studies of 2-(4-fluorophenylamino)-5-(2,4-dihydroxyphenyl)-1,3,4-thiadiazole (FABT), as one of the

most promising derivatives from the N-substituted 2-amino-5-(2,4-dihydroxyphenyl)-1,3,4-thiadiazole set, have been continued.
The tested compound inhibited proliferation of tumor cells derived from cancers of nervous system (medulloblastoma/rhabdosar-
coma, neuroblastoma, and glioma) and peripheral cancers including colon adenocarcinoma and lung carcinoma. The anticancer
effect of FABT was attributed to decreased cell division and inhibited cell migration. Furthermore, in anticancer concentrations it
exerted a trophic effect in neuronal cell culture and had no influence on viability of normal cells including astrocytes, hepatocytes,
and skin fibroblasts. Moreover, a prominent neuroprotective activity of FABT was observed in the neuronal cultures exposed to neu-
rotoxic agents like serum deprivation and glutamate. To determine probability of tautomeric transition and indicate potential sites of
interactions of FABT molecule with the receptor, quantum-chemical calculations with the ab initio Hartree–Fock model were made.
 2007 Elsevier Ltd. All rights reserved.

1. Introduction type of derivatives a different mechanism of action is

Despite significant progress achieved in anticancer ther-
apy, the management of malignancies in humans still
constitutes a major challenge for contemporary medi-
cine.1–4 Chemotherapy very often causes severe side
effects, which are in part a consequence of destruction
of normal cells. It was revealed that commonly used
anticancer drugs cause significant toxicity in nervous
system and are responsible for neurological side
effects.5–9 It is of crucial importance that anticancer
drugs display antiproliferative activity in tumor cells
without affecting normal tissues. Therefore, taking all
the above-mentioned evidence into account, the develop-
ment of novel chemotherapuetics and effective anticancer
strategies is eagerly being pursued.
2-Amino-1,3,4-thiadiazole and its derivatives are well
known as compounds of a wide range of anticancer
activity,10–15 including in vivo conditions.16–18 For this
Keywords: 2-Amino-1,3,4-thiadiazole; Anticancer activity; Neuropro-
tective activity; Molecular descriptors.
* Corresponding author. Tel.: +48 81 4456816; fax: +48 81 5333752;
e-mail: joanna.matysiak@ar.lublin.pl
assigned, depending on the type of modification of
1,3,4-thiadiazole ring.19–21 The action connected with
the apoptotic mechanisms and angiogenesis, which is a
crucial step in the tumorigenesis, seems to be very
promising in anticancer therapy.22,23 It was found that
(E,E)-2,5-bis[4-(3-dimethylaminopropoxy)styryl]-1,3,4-
thiadiazole induced the early-phase apoptosis in human
non-small lung cancer A549 cells via the Bcl-XL down-
regulation, and that of the late-phase through up-regula-
tion of Bax expression as well as inhibition of Akt/
protein kinaze B (PKB). In addition, the compound
showed equivalent anti-angiogenic activity in the nude
mice angiogenesis model.24
In our previous publication we described synthesis and
in vitro antiproliferative activity against some human
cancer cell lines of compounds of 2-amino-5-(2,4-
dihydroxyphenyl)-1,3,4-thiadiazole set.25,26 The highest
antiproliferative effect for N-halogenphenyl derivatives
was found.26 Therefore, we decided to extend studies
on this type of compounds.
We herein report the further in vitro evaluation of
2-(4-fluorophenyloamino)-5-(2,4-dihydroxyphenyl)-1,3,

0968-0896/$ - see front matter  2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.02.041
3202 W. Rzeski et al. / Bioorg. Med. Chem. 15 (2007) 3201–3207

4-thiadiazole (FABT). The anticancer profile and neuro-

protective activity of the compound are described. To
determine probability of tautomeric transition and indi-
cate potential sites of interactions of FABT molecule
with the receptor, quantum-chemical calculations were
carried out with the ab initio Hartree–Fock model.

2. Results and discussion

2.1. Biology

The antiproliferative effect of FABT (Fig. 1) was as-

sessed in a range of human tumor cell lines. Tumor cells
derived from cancers of nervous system (medulloblas-
toma/rhabdosarcoma, neuroblastoma, and glioma) and
peripheral cancers including colon adenocarcinoma
and lung carcinoma were tested. The cells were exposed
to either culture medium (control) and FABT
(5–100 lM) for 96 h. Proliferation of all tumor cell cul-
tures was decreased in the cultures exposed to FABT
(Fig. 2a) in a concentration-dependent fashion as mea-
sured by means of the MTT assay. Threshold concentra-
tions of FABT required to elicit antiproliferative effect
were as low as 5 lM (HT-29), 10 lM (A549, TE671),
and 25 lM (SK-N-AS, C6). The effect of FABT on
Figure 2. (a) Antiproliferative effects of FABT in cancer cells. FABT
tumor cells was attributed to decreased cell division as
exerts a concentration (log c)-dependent antiproliferative effect in a
determined by measurements of incorporation of BrdU range of tumor cell lines. Cells were exposed to either culture medium
during the DNA synthesis (Fig. 3). It is of crucial impor- alone (control) and FABT (5–100 lM) for 96 h, and viability was
tance that anticancer drugs display antiproliferative measured by means of MTT assay. IC50: SK-N-AS, 54.7 lM; TE671,
activity in tumor cells without affecting normal cells. 26.0 lM; HT-29, 33.1 lM; A549, 22.8 lM; C6, 27.3 lM. SK-N-AS,
We have shown that normal cells were resistant to human neuroblastoma; C6, rat glioma; TE671, human rhabdomyo-
FABT up to 100 lM. Moreover, in the neuronal culture sarcoma–medulloblastoma; A549, human lung carcinoma; HT-29,
it showed a prominent neurotrophic activity (Fig. 2b). human colon adenocarcinoma; Fao, rat hepatocytes. (b) Effect of
Chemotherapy very often induces severe side effects, FABT on normal cells’ viability. Rat cortical neurons, astrocytes, and
which are in part a consequence of destruction of nor- hepatocytes (Fao) were exposed to either culture medium alone
(control) and FABT (5–100 lM) for 48 h, and the viability was
mal cells.6 To evaluate the cytotoxic effect on normal
measured by means of MTT assay. The data represent mean
cells, human skin fibroblasts (HSF) were subjected to normalized optical densities ±SEM of 5–8 trials and were analyzed
increasing doses of FABT (5–250 lM). LDH assay re- by means of linear regression.
vealed that FABT produced low cytotoxicity in normal
HSF cell culture. The significant LDH release after
24 h exposure appeared at 100 lM (not shown). These
data can suggest a beneficial side-effect profile of FABT.

The ability of tumor cells to migrate is one of the mark-

ers of tumor metastatic potential. To evaluate the effect
of FABT on tumor cell motility, glioma (C6) and lung
carcinoma (A549) cells were exposed to culture medium
alone and 5 lM of FABT for 24 h. The wound assay
revealed that in the cultures exposed to FABT, signifi-
cantly fewer cells migrated to the wound area
(Fig. 4a). The micrographs showing wound, cell migra-
tion in control culture, and inhibited glioma C6 cells
Figure 3. The antiproliferative effect of FABT was attributed to
migration following FABT exposure are presented in decreased cell division. Human lung carcinoma (A549) cells and rat
glioma (C6) cells were grown in culture medium only (control) and in
the presence of FABT (5–50 lM) for 48 h. BrdU incorporation was
used as a marker of cell division. The data represent mean normalized
S optical densities ±SEM of 5–8 trials and were analyzed by means of
HO NH F linear regression. IC50: C6, 23.5 lM; A549, 13.7 lM.
N N
OH
Figure 4b–d. The influence on tumor cell locomotion
Figure 1. Chemical structure of 2-(4-fluorophenylamino)-5-(2,4- can suggest an antimetastatic potential of tested amino-
dihydroxyphenyl)-1,3,4-thiadiazole (FABT). thiadiazole derivative.
 W. Rzeski et al. / Bioorg. Med. Chem. 15 (2007) 3201–3207 3203

Figure 4. Effect of FABT on migration of tumor cells. Wounded monolayers of tumor cells (C6, A549) were incubated for 24 h alone or in the
presence of FABT (5 lM). Tumor cells, which migrated to the wound area, were counted (a). Micrographs show wound assay of glioma C6 cells;
wound (b), cell migration after 24 h in control culture (c) and following exposure to 5 lM FABT (d). Magnification 40·. The results are expressed as
the mean number of cells migrated per field of the wound area ±SEM of 50 measurements; ***, +++ at least p < 0.001 versus control, Student’s t test.

Glutamate is the major neurotransmitter in the mamma-

lian nervous system.27 Abnormal glutamate signaling
has been associated with neurological and psychiatric
disorders including stroke, epilepsy, and neurodegenera-
tive diseases, such as Alzheimer’s, and Parkinson’s dis-
ease.28–30 In order to characterize the neuroprotective
effects of tested compound, rat cortical neurons were ex-
posed to the neurodegenerative agents like serum depri-
vation (SD) and glutamate (500 lM) alone and
combined with FABT (10 and 25 lM). Both applied
agents induced a prominent neurotoxicity which was
further ameliorated by coexposure with FABT in a
dose-dependent manner (Fig. 5). These preliminary data
suggest that FABT exerts a prominent neuroprotective
activity. Of note is the fact that concentrations of FABT
required to elicit neuroprotection strongly influence tu-
mor cell proliferation. In the nervous system, glutamate Figure 5. Neuroprotective effects of FABT. Neurodegeneration was
induced by exposure of neuronal cell culture to serum deprivation (SD)
activates ionotropic and metabotropic receptors. Gluta-
and glutamate (500 lM). FABT (10, 25 lM) significantly ameliorates
mate antagonists, which selectively block glutamate in vitro induced neurotoxicity. Columns represent mean normalized
receptors, were demonstrated to have neuroprotective optical densities ±SEM of 4–8 trials. Statistical comparisons were
properties.31 Recently, in a series of experiments we performed between the results obtained with control, the neurotoxic
have described that these compounds elicit anticancer agent alone, and those obtained in combination with FABT (+FABT).
effects in vitro and in vivo.32,33 The answer to the ques- ***p < 0.001, +p< 0.05, +++p < 0.001 for exposure to SD; ###p < 0.001,
&&&
tion whether FABT exerts glutamate antagonist proper- p < 0.001 for exposure to glutamate; Student’s t test.
ties will be a subject of further studies. Also, the in vivo
studies and characterization of molecular mechanisms activity remain to be done in order to evaluate its poten-
involved in FABT anticancer and neuroprotective tial application as a new drug.
3204 W. Rzeski et al. / Bioorg. Med. Chem. 15 (2007) 3201–3207

2.2. Computational studies

Literature reports indicate existence of tautomeric forms

of N-substituted 2-amino-1,3,4-thiadiazoles both in
solutions and solid phase which can be of significant
importance for biological activity.34 The direction of
equilibrium shift (Fig. 6) depends largely on substitution
type of thiadiazole ring.

To determine probability of tautomeric transition and

indicate potential sites of interactions of FABT molecule
as one of the most active analogs from N-substituted
2-amino-5-(2,4-dihydroxyphenyl)-1,3,4-thiadiazole set,
the quantum-chemical calculations were carried out
with the ab initio Hartree–Fock model at the 6-31G**
basis set. The determined values of HF energy (EHF)
of isolate molecule of both forms indicate thermody-
namic preferences of the amine form A (Table 1). The
additional indication is its smaller surface area, volume,
and differences of heat formation (HF) and energy solva-
tation (ES) for both species. Existence of the imine form
(B) for FABT was not confirmed in the determination
conditions using spectroscopic methods. In spite of a
relatively high energetic barrier of the tautomeric transi-
tion in the environmental conditions, after taking Figure 7. Molecular electrostatic potentials (MEPs) showing the most
mainly solvatation process into account, the equilibrium positive potential (deepest blue color), the most negative potential
(deepest red color), and the intermediate potential (intermediate
coexisting imine form (B) cannot be excluded.
shades) regions. On MEPs the electrostatic potential profile at
20 kcal/mol was superimposed; (a) amine and (b) imine forms of
Molecular potential density distribution for amine struc- FABT.
ture (A) (Fig. 7) shows the most negative potential on
nitrogen atoms of thiadiazole ring but the most positive
on amine hydrogen atom. This may indicate capability
of hydrogen bond formation with share of nitrogen atoms are confirmed by position of resonance signals in
atoms of the ring or through hydrogen atom of –NH- the 13C NMR.25,26
moiety. Negative potential is located on oxygen and
fluorine atoms additionally. This is in agreement with Assuming existence of tautomeric imine form (B) the
LUMO and HOMO orbitals distribution of thiadiazole analogous fragment of the molecule should be regarded
moiety (Fig. 8). LUMO involves sulfur and second as a potential site of interactions, whereby due to proton
carbon atom mainly, indicating the region reacting with transfer, only one of nitrogen atoms would retain its
potential biological nucleophiles. HOMO is located on previous properties of proton acceptor (N-4 of thiadiaz-
nitrogen atoms (including amine atom) and represents ole ring), but two would change their functions into re-
the space of the greatest electron density. A large elec- verse ones (Fig. 7b). Additionally, the electron gap on
tron gap on C-2 compared to C-5 of 1,3,4-thiadiazole C-2 ring disappears. The charges of both carbon atoms
ring determined from the electrostatic potential distribu- of 1,3,4-thiadiazole ring become equal (Table 1).
tion can also be essential in interactions with the recep-
tor (Table 1). Experimentally large differences in Compound activity can be additionally intensified by the
electron density distribution for both mentioned carbon fragment of 2,4-dihydroxyphenyl, donor or acceptor of
protons for hydroxyl groups. The ortho-position group
of amine form (A) is involved in the intramolecular
hydrogen bond which stabilized the planar conforma-
S R S tion. This is confirmed by numerous literature reports
HO N HO N R
on various analogs as well as by X-ray structural inves-
N N H N N
OH OH H
tigations by our team.35 Besides direct interactions with
amine tautomer (A) imine tautomer (B)
the receptor, hydroxyl groups probably affect many
other factors like transport, toxicity, essential for biolog-
Figure 6. Tautomeric equilibrium for 2-amino-1,3,4-thiadiazoles. ical activity. Large potential accumulated on fluorine

Table 1. Structural, electronic, and thermodynamic properties of two species of FABT

Form EHF (kJ/mol) Volume (Å3) Area (Å2) ELUMO (eV) EHOMO (eV) HF (kcal/mol) ES (kJ/mol) qC-2 (e) qC-5 (e)
Amine (A) 3533230.3 299.56 307.68 2.426 7.862 11.26 17.39 0.70 0.31
Imine (B) 3533192.5 302.43 314.03 2.870 8.149 9.92 12.47 0.55 0.57
 W. Rzeski et al. / Bioorg. Med. Chem. 15 (2007) 3201–3207
Figure 8. LUMO and HOMO isosurfaces for the amine form of
FABT. Different surface colors represent opposite signs of the
wavefunction. The structures are shown with a tube rendering;
nitrogen atoms are in blue and sulfur atom is in green.
atom characteristic of both tautomeric forms can also
play a significant role (Fig. 7).
The analysis indicates that independent of the form
(though the amine form is probably predominant in the
case of FABT), the moiety of three nitrogen atoms com-
bined in the amino-five-membered system by means of
sulfur atom is responsible for activity. Sulfur atom in a
five-membered ring can affect relatively high aromatiza-
tion of the system with relatively larger accessibility of
free orbitals compared to analogous arrangements of
atoms in the isostructural rings and intensifies electron
density on nitrogen atoms due to greater tendency to
pass into the electropositive state compared to other het-
eroatoms. It probably decides also about the required
stericity, that is, bond length, angle size. Activity of this
pharmacophore is, on one hand, intensified by the 2,4-
dihydroxyphenyl substituent and on the other hand, by
the para-fluorophenyl ring of exogenous nitrogen atom.
3. Conclusion
To sum up, we described herein the in vitro anticancer
and neuroprotective activity of FABT. In a series of
in vitro experiments we have shown that a new 2-ami-
no-1,3,4-thiadiazole derivative elicits prominent antican-
cer effects in a range of tumor cell cultures. At the same
time, the tested compound was not toxic to normal cells.
3205
Moreover, it possesses a prominent neuroprotective
activity. Quantum-chemical calculations confirm the
function of aminothiadiazole moiety as pharmacophore
of this type of activity, intensified by the 2,4-dihydroxy-
phenyl substituent, independently of other properties of
this benzenodiole moiety, and on the other hand, by the
para-fluorophenyl substituent.
4. Materials and methods
4.1. Preparation of FABT
FABT (Fig. 1) was obtained from 4-(4-fluorophenyl)-3-
thiosemicarbazide (Lancaster) and sulfinylbis(2,4-dihy-
droxythiobenzoyl) in the endocyclization process.26
Mp: 279–280 C; 1H NMR (200 MHz, DMSO-d6, d):
10.85 (s, 1H, 2-COH), 10.27 (s, 1H, 4-COH), 9.92 (s,
1H, NH); IR (KBr, cm 1): 3400, 3259, 3216 (OH,
NH), 1629 (C@N), 1590 (C@C), 1231 (C–OH), 1183,
1134 (C–F), 1052 (N@C–S–C@N), 677 (C–S–C); EI-
MS (m/z, %): 303 (M+, 100) Anal. Calcd for
C14H10FN3O2S (303.32): C, 55.44; H, 3.22; N, 13.85.
Found: C, 55.61; H, 3.23; N, 13.79.
4.2. Cell lines
Human rhabdomyosarcoma/medulloblastoma (TE671),
human neuroblastoma (SK-N-AS), and rat hepatocytes
(Fao) were obtained the from European Collection of
Cell Cultures (Center for Applied Microbiology and
Research, Salisbury, UK). Human Caucasian lung car-
cinoma (A549) and human colon adenocarcinoma
(HT-29) were obtained from the Institute of Immunol-
ogy and Experimental Therapy (Polish Academy of
Sciences, Wrocław, Poland). Rat glioma (C6) was
obtained from the Department of Neonatology, Cha-
rite-Virchow Clinics, Humboldt University, Berlin,
Germany. Human skin fibroblasts (HSF) were a labora-
tory strain obtained by the outgrowth technique from
skin explants of young persons.
The following culture media purchased from Sigma (Sig-
ma Chemicals, St. Louis, MO, USA) were applied:
DMEM (HT-29, C6, HSF), 1:1 mixture of DMEM
and Nutrient mixture F-12 Ham (TE671, SK-N-AS),
2:1 mixture of DMEM and Nutrient mixture Ham’s
F-12 (A549), and Ham’s F-12K (Fao). All media were
supplemented with 10% FBS (Life Technologies,
Karlsruhe, Germany), penicillin (100 U/mL) (Sigma),
and streptomycin (100 lg/mL) (Sigma). The cultures
were kept at 37 C in humidified atmosphere of 95%
air and 5% CO2.
4.3. Neuronal cell culture
The neuronal cell culture was prepared from cortices of
18-day-old Wistar rat fetuses as previously described.36
The tissue was pooled into ice cold glucose (33 mM)
Hanks’ Balanced Salt Solution (HBSS), cut into small
pieces, and incubated for 30 min at 37 C with 0.25%
trypsin-EDTA solution. A single cell suspension was ob-
tained by gentle pipetting of the cortex fragments in the
3206 W. Rzeski et al. / Bioorg. Med. Chem. 15 (2007) 3201–3207
presence of 10% FBS and 0.01% DNase I (Sigma). The
cells were then sieved through the 40 lM cell strainer
(Falcon, Becton Dickinson Labware, Franklin Lakes,
New Jersey, USA), centrifuged at 800 rpm for 10 min,
and plated at 5 · 105 cells/mL density on poly-L -lysine
(MW 70,000–150,000) coated 96-multiwell plates (Nunc,
Roskilde, Denmark). The culture medium consisted of
B-27 supplemented Neurobasal Medium (Life Technol-
ogies) which eliminates glial growth, 0.5 mM L -gluta-
mine, and 1% of antibiotic-antimycotic solution (Life
Technologies). For the initial plating the culture med-
ium was supplemented with 25 lM glutamate. Incuba-
tion was carried out at 37 C in humidified 95% air
and 5% CO2 atmosphere. The culture medium was chan-
ged every 3 days, until the culture reached 14 days. Neu-
ronal identity was confirmed by positive staining with
the mouse anti-neuron specific enolase cc monoclonal
antibody (Chemicon International, Inc., Single Oak
Drive, Temecula, CA, USA). Antibody detection and
antigen visualization were performed by using Streptavi-
din HRP Kit (STAR 2004) (Serotec Ltd, Oxford, UK).
4.4. Glial cell culture
The primary mixed glial cell culture was initially estab-
lished according to the same protocol used for neurons.
The cells were inoculated (15 · 106) into 75 cm2 TC
flasks and left for 48 h at 37 C in 5% CO2 incubator.
Culture medium (1:1 mixture of DMEM and Ham
F-12 nutrient mixture supplemented with 10% FBS,
100 U/ml penicillin, and 100 lg/mL streptomycin) was
changed daily until the culture reached confluency
(7–10 days). The flasks were shaken overnight in the
orbital shaker at 210 rpm in order to remove less adher-
ent cells (neurons, microglia, and oligodendroglia).
Following this shaking procedure, the culture became
enriched with flat cells displaying typical astrocyte
morphology. Immunostaining with a primary antibody
for glial fibrillary acidic protein (GFAP, polyclonal,
DAKO, Denmark) revealed that astrocytes accounted
for 95% of the cells in the culture.
4.5. Viability assay
Tumor cells were plated on 96-well microplates (Nunc)
at a density of 0.5 · 104 (C6), 1 · 104 (TE671, A549),
and 3 · 104 (HT-29, SK-N-AS). Next day the culture
medium was removed and the cells were exposed to
serial dilutions of FABT in a fresh medium. Cell prolif-
eration was assessed after 96 h by using the MTT meth-
od (Cell proliferation kit I, Boehringer Mannheim,
Germany) in which the yellow tetrazolium salt (MTT)
is metabolized by viable cells to purple formazan
crystals. Tumor cells were incubated for 4 h with MTT
solution (5 mg/mL). Formazan crystals were solubilized
overnight in SDS buffer (10% SDS in 0.01 N HCl) and
the product was quantified spectrophotometrically by
measuring absorbance at 570 nm wavelength using
E-max Microplate Reader (Molecular Devices Corpora-
tion, Menlo Park, CA, USA).
Normal cells were plated on 96-well microplates (Nunc)
at a density of 5 · 105 (neurons), 2 · 105 (astrocytes),
and 2.5 · 105 (Fao). Next day the culture medium
was removed and the cells were exposed to FABT
(5–100 lM) for 48 h. Astrocytes and Fao cells were
incubated in the medium containing 2% of FBS.
Viability was assessed by means of MTT method.
In neuroprotection experiments, neurons were exposed
to serum deprivation (SD—Neurobasal medium with-
out B-27 supplement) and glutamate (500 lM) alone
and combined with FABT (10 and 25 lM). Cell viability
was assessed after 48 h by means of MTT method.
4.6. Proliferation assay
Cells were plated on 96-well microplates (Nunc) at a
density of 1 · 104 (C6) and 2 · 104 (A549). Next day
the culture medium was removed and the cells were
exposed to serial dilutions of FABT in a fresh medium.
Cell proliferation was quantified after 48 h by measure-
ment of BrdU incorporation during DNA synthesis
(Cell Proliferation ELISA BrdU, Roche Diagnostics
GmbH, Penzberg, Germany). Tumor cells were incu-
bated with 10 lM BrdU for 2 h. The cells were subse-
quently incubated with the FixDenat solution for
30 min and then exposed to monoclonal anti-BrdU anti-
bodies conjugated to peroxidase. Color reaction was
developed by adding the TMB substrate solution and
terminated by addition of 1 M H2SO4. The absorbance
was measured at 450 nm wavelength using E-max
Microplate Reader.
4.7. Cytotoxicity assay
A cytotoxicity detection kit based on measurement of
lactate dehydrogenase (LDH) activity was applied
(Tox-7, Sigma). The assay is based on the reduction of
NAD by the action of LDH released from damaged
cells. The resulting NADH is utilized in stoichiometric
conversion of a tetrazolium dye. The resulting colored
compound is measured spectrophotometrically. Human
skin fibroblasts (HSF) were plated on 96-well micro-
plates at a density of 1 · 105. Next day the culture med-
ium was removed and the cells were subjected to the
tested compound (5–100 lM) diluted in a fresh culture
medium with reduced amount of FBS (2%). Culture
supernatants were collected after 24 h and incubated
with substrate mixture for 30 min at room temperature
in the dark. At the end, the reaction was terminated
by the addition of 1 N HCl and the color product was
quantified spectrophotometrically at 450 nm wavelength
using E-max Microplate Reader.
4.8. Cell migration assessment
Tumor cell migration was assessed in the wound assay
model. Tumor cells (C6, A549) were plated at 1 · 106
cells on 4-cm culture dishes (Nunc). Next day, the cell
monolayer was scratched by the pipette tip (P300), the
medium and dislodged cells were aspirated, and the
plates rinsed twice with PBS. Next, the fresh culture
medium was applied and the number of cells migrated
into the wound area after 24 h was estimated in the
control and the cultures treated with FABT (5 lM).
 W. Rzeski et al. / Bioorg. Med. Chem. 15 (2007) 3201–3207
The plates were stained with the May-Grünwald–Giem-
sa method. The observation was performed in Olympus
BX51 System Microscope (Olympus Optical CO., LTD,
Tokyo, Japan) and the micrographs were prepared in
analySIS software (Soft Imaging System GmbH, Mün-
ster, Germany). Cells migrated to the wound area were
counted on micrographs and results expressed as a mean
cell number migrated to the selected 50 wound areas
taken from four micrographs.
4.9. Computational methods
The compound was built with a standard bond length
and angles using the PC SPARATN Pro Ver 1.08 molec-
ular modelling program.37 The energy was minimized by
the molecular mechanic methods and then by the Har-
tree–Fock method at 6-31G**. Charge of atoms from
the electrostatic potential distribution was determined.38
Heat of formation was calculated using the PM3
method.
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